26th Annual Computational Neuroscience Meeting (CNS*2017): Part 3 - Meeting Abstracts - Antwerp, Belgium. 15–20 July 2017

This work was produced as part of the activities of FAPESP Research,\ud Disseminations and Innovation Center for Neuromathematics (grant\ud 2013/07699-0, S. Paulo Research Foundation). NLK is supported by a\ud FAPESP postdoctoral fellowship (grant 2016/03855-5). ACR is partially\ud supported by a CNPq fellowship (grant 306251/2014-0)

A pan-genotypic Hepatitis C Virus NS5A amplification method for reliable genotyping and resistance testing

Background: Chronic infection with the Hepatitis C Virus (HCV) is associated with the risk of progressive liver disease. Although, HCV treatment options and viral cure rates have tremendously increased over the last decade, all currently licensed combination therapies contain inhibitors of the replication complex NS5A. Resistance-associated substitutions (RAS) in NS5A can limit the efficacy of therapy; however, resistance testing is routinely not recommended for all patients. Notably, pan-genotypic combinations have been approved, however the correct identification of the HCV genotype is still required for treatment decisions and is a good predictor for treatment success. Objective: The aim of this study was the establishment of a pan-genotypic NS5A amplification method for reliable genotyping and simultaneous resistance testing in a fast and cheap routine diagnostic setup. Study design: Pan-genotypic degenerated nested PCR primer were designed and tested in 262 HCV-patients. The collection included samples from genotypes 1-7 and the median viral load was 1.07x10(6) IU/ml (range 248-21x10(6) IU/ml). Results: Amplification of the expected 747bp fragment was successful in 257 of 262 (98.1%) samples including samples < 1000 IU/ml. The direct comparison of the genotype information obtained with core sequencing to those obtained by NS5A prediction showed high concordance (97.3%) and discrepancies occurred only for relatively rare subtypes. Resistance analysis using Geno2Pheno([HCV]) showed NS5A-RAS in 23 of 257 (8.9%) of samples. Conclusions: We successfully developed a routine diagnostic method for pan-genotypic amplification of NS5A. This amplicon can be used for simultaneous genotyping and resistance testing for enhancing and improving routine HCV diagnostic

A genotype independent, full-genome reverse-transcription protocol for HCV genotyping and resistance testing

Background: HCV treatment options and cure rates have tremendously increased in the last decade. Although a pan-genotype HCV treatment has recently been approved, most DAA therapies are still genotype specific. Resistance-associated variants (RAVs) can limit the efficacy of DAA therapy and are associated with increased risk for therapy failure. With the approval of DAA regimens that recommend resistance testing prior to therapy, correct assessment of the genotype and testing for viruses with RAVs is clinically relevant. However, genotyping and resistance testing is generally done in costly and laborious separate reactions. Objective: The aim of the study was to establish a genotype-independent full-genome reverse transcription protocol to generate a template for both genotyping and resistance testing and to implement it into our routine diagnostic setup. Study design: The complete HCV genome was reverse transcribed with a pan-genotype primer binding at the 3' end of the viral RNA. This cDNA served as template for transcription of the genotyping amplicon in the core region as well as for the resistance testing of NS3, NS5A, and NS5B. Results: With the established RT-protocol the HCV core region was successfully amplified and genotyped from 124 out of 125 (99.2%) HCV-positive samples. The amplification efficiency of RAV containing regions in NS3, NS5A, NS5B was 96.2%, 96.6% and 94.4%, respectively. Conclusions: We developed a method for HCV full-genome cDNA synthesis and implemented it into a routine diagnostic setup. This cDNA can be used as template for genotyping amplicons covering the core or NS5B region as well as for resistance testing amplicons in NS3, NS5A and NS5B

Determination of Drug Resistance and Virus Typology in HIV-1-Positive Pediatric Patients in Istanbul, Turkey

The aim of the study was to determine the prevalence of drug resistance of HIV-1 in pediatric patients from Istanbul, Turkey. Genotypic drug resistance testing revealed transmission of drug resistance from mother to child in 20%. Due to rising numbers of children with HIV-1, baseline resistance testing is recommended for Turkey. (C) 2014 S. Karger AG, Base

Detection of a genetic footprint of the sofosbuvir resistance-associated substitution S282T after HCV treatment failure

Background: The major resistance-associated substitution for sofosbuvir (S282T) in HCV NS5B causes severe viral fitness costs and rapidly reverts back to prototype in the absence of selection pressure. Accordingly, resistance against sofosbuvir is rarely detected even in patients after treatment failure. Case presentation: We report a case of a GT3a infected patient with viral breakthrough under SOF/DCV therapy. At the time of breakthrough the RAS S282T was predominant in NS5B and then rapidly disappeared during follow-up by week 12 after treatment. Interestingly, despite only serine was encoded in position 282 during follow-up, two distinct genetic pathways for reversion were detectable. In 31% of the quasispecies the original codon for serine was present whereas in the majority of the quasispecies an alternative codon was selected. This alternative codon usage was unique for all GT3a isolates from the HCV database and remained detectable as a genetic footprint for prior resistance selection at the RNA level for at least 6 months. Conclusions: Comparative analyses of viral sequences at the codon level before and after DAA treatment may help to elucidate the patient's history of resistance selection, which is particularly valuable for highly unfit substitutions that are detectable only for a short period of time. If such codon changes increase the risk of re-selection of resistance upon a second exposure to SOF remains to be addressed

Molecular epidemiology of HIV in a cohort of men having sex with men from Istanbul

In Turkey, the first HIV/AIDS case was reported in 1985. Since then the number of persons with HIV infection has increased, HIV is getting a public health problem. The aim of this study was to determine HIV-1 subtype diversity, drug resistance and gag cleavage site mutations among 20 HIV-infected men having sex with men from Istanbul, Turkey. The most prevalent subtype was found to be subtype B (50 %), but also the non-B subtypes A1, C and CRF02_AG, CRF03_AB and CRF06_cpx were found. Resistance-associated mutations were found in 6 patients (30 %) with 2/6 patients being therapy-experienced and 4/6 therapy-na < ve at the time-point of analysis. In these patients, the nucleoside reverse transcriptase inhibitor (NRTI)-associated resistance mutations M41L, T215C, V75I, T69N, the non-NRTI associated mutations V106I, E138A, K103N and the protease inhibitor associated mutations Q58E and V82I were detected. Two virus strains also presented Gag cleavage site mutations. With increasing numbers of HIV-infected Turkish patients that require anti-retroviral treatment, HIV-1 drug-resistance testing is strongly recommended in order to choose the most active drug combination for therapy to achieve better clinical outcomes

Dynamics of BKPyV reactivation and risk of hemorrhagic cystitis after allogeneic hematopoietic stem cell transplantation

ObjectivesAim of this retrospective study was to analyze the dynamics of BKPyV reactivation in allogeneic hematopoietic stem cell transplant recipients in order to identify patients with higher risk to develop BKPyV-associated hemorrhagic cystitis (BKPyV-associated HC). MethodsThe study included 58 allo-HSCT recipients from the University Hospital of Cologne detected BKPyV positive by real-time PCR between 2009 and 2015. For correlative analysis, the first detected BKPyV-DNA load in urine and in plasma as well as the onset and severity of HC following the first day of conditioning regimen was considered. Phylogenetic analysis of BKPyV isolates was performed. ResultsIn 25 of 58 patients, BKPyV-DNA was detected in urine only (group U), whereas 33 patients developed additional viremia (group P). A chronologic sequence viruria-viremia-HC was identified. Viral load of >10(6) copies/mL at first viruria and evidence of viremia after 45days from the start of conditioning represented risk factors for the onset of HC. Molecular characterization revealed a non-stereotypic distribution of viral subtypes across groups U and P. ConclusionsMonitoring of BKPyV-DNA by real-time PCR after initiation of conditioning, regularly performed in clinical practice, can be a crucial tool for the early identification of patients with higher risk of BKPyV-associated HC

The SnoB study: frequency of baseline raltegravir resistance mutations prevalence in different non-B subtypes

The SnoB study analysed the variability of the integrase (IN) gene of non-B viruses from treatment-naive patients to determine whether non-B subtypes carry natural resistance mutations to raltegravir (RAL). Plasma viral RNA from 427 patients was gained, and IN sequences were subtyped and screened for subtype-specific highly-variable residues. Seven viruses of different subtypes were phenotypically tested for RAL susceptibility; 359/427 samples could be sequenced. One hundred and seventy samples (47%) were classified as non-B subtypes. No primary RAL resistance-associated mutations (RRAMs) were detected. Certain secondary mutations were found, mostly related to specific non-B subtypes. L74 M was significantly more prevalent in subtype 02_AG, T97A in A and 06_cpx, V151I in 06_cpx, and G163R in 12_BF. Various additional mutations were also detected and could be associated with the subtype too. While K156 N and S230 N were correlated with B subtype, V72I, L74I, T112I, T125A, V201I and T206S were more frequent in certain non-B subtypes. The resistance factors (RF) of 7 viral strains of different subtypes ranged from 1.0 to 1.9. No primary or secondary but subtype-associated additional RRAMs were present. No correlation between RF and additional RRAMs was found. The prevalence of RRAMs was higher in non-B samples. However, the RFs for the analysed non-B subtypes showed lower values to those reported relevant to clinical failure. As the role of baseline secondary and additional mutations on RAL therapy failure is actually not known, baseline IN screening is necessary

Detection and characterization of enteroviruses and parechoviruses in healthy people living in the South of Cote d'Ivoire

Background: Human enteroviruses (EVs) and parechoviruses (HPeVs) belong to the family Picornaviridae. Although most EV and HPeV infections remain asymptomatic, both pathogens can cause a wide spectrum of clinical manifestations ranging from respiratory or gastrointestinal symptoms to myocarditis, neonatal sepsis, and infections of the central nervous system. Objectives: Aim of the present study was to investigate the spectrum of EVs and HPeVs in apparently healthy adults and children living in the South of Cote d'Ivoire. Study design: The study included 105 stool samples obtained from healthy individuals aged 0-53 years between June 2013 and December 2014 in the Sud-Como region of Cote d'Ivoire. After collection and shipment to Germany, the samples were analyzed by real-time PCR for the presence of EVs and HPeVs RNA. Molecular typing and virus isolation of all samples were performed.e Results: Out of 105 samples, 24 (22.8%) were EV positive and six (5.2%) were HPeV positive. Twenty-one EV positive samples could be characterized with serotypes belonging to EV group A-C, while three could not be further specified. Interestingly, several rarely described serotypes were identified, e.g., EV-C99, EV-B93, EV-C116, and EV-A119. Typing of HPeV positive samples resulted in HPeV-1 and -5 detections, while one isolate could not be assigned to the known HPeV types. Conclusions: This study showed a large variety of EV strains in healthy people in the South of Cote d'Ivoire and provided the first available data about HPeV infections in a sub-Saharan African country. (C) 2015 Elsevier B.V. All rights reserved